PROJECT SUMMARY Genetic factors play an important role in the pathogenesis and progression of Alzheimer's disease (AD). One hallmark of AD is the development of amyloid (A) neuritic plagues and fibrils, which is believed to play a central role in neuronal cell loss. Most people of advanced age develop amyloid plagues and fibrils in their brains, but only a subset of the aging population develops clinical signs of AD. This finding suggests the existence of gene regulatory pathways controlling disease development and potentially disease severity. Protective mechanisms against AD appear to exist, but have not been characterized at the genetic or molecular level. Herein, we propose to screen for genes/pathways in mammalian cells that confer protection against AD by using resistance to A cytotoxicity as a surrogate. Our laboratory has generated a library of mouse mutant embryonic stem (ES) cells, comprising 42,000 independent mutants mutagenized by piggyBac (PB) transposon-mediated gene entrapment. We will utilize A42 oligomers for the selection of resistance mutants in a forward genetic screen via a high-throughput ES cell platform. This novel approach will allow for an unbiased interrogation of the genome, potentially revealing novel functional pathways protective against AD, which may provide new insights into therapeutic targets for AD treatment. Our long-term plan is to screen the entire library (42,000 mutants) to isolate A resistance mutants and identify the underlying genes and pathways. In this application, we will establish the necessary techniques required to select A resistance and to identify resistance genes; a pilot screening will be conducted as a proof of principle. A affects neurons, so we will differentiate our mutant ES cells into neurons, followed by selection for A resistance in 96-well plates. Mutants showing a significant increase in survival in the presence of A will be isolated for extensive characterizations, including the identification of the mutated gene(s), the nature of the mutational events (null, partial loss of function, or gain of function), and the confirmation of the causal relationship between the disrupted gene and the A resistance phenotype. Once those genes are identified, their functions can also be confirmed in human neurons by engineering the mutations using CRISPR. This study pursues an innovative approach encompassing the use of stem cells, a transposon, and forward genetics to identify genes/pathways protective against AD. Furthermore, the ES cells carrying AD-protective mutations may be used in subsequent experiments to generate the corresponding mice for in vivo studies.